Molecular prevalence and phylogenetic analysis of hemotropic Mycoplasma species in cats in different regions of Iran

Background Hemotropic Mycoplasma species (hemoplasmas) cause hemolytic anemia in cats worldwide and are recognized as emerging zoonotic pathogens. There is no comprehensive study on the prevalence and species diversity of hemoplasmas in domestic cat populations in different regions in Iran. Thus, the aims of the present study were to provide data on the prevalence and molecular characterization of hemotropic Mycoplasma species in apparently healthy cats from six Iranian provinces with different climates. In addition, potential risk factors associated with hemoplasmosis in cats were assessed. Results Mycoplasma spp. DNA was detected in the blood of 56 / 361 cats (15.5%) using genus-specific PCR. Further examinations with species-specific PCR and Sanger sequencing showed that 38 cats (10.5%) tested positive for Candidatus Mycoplasma haemominutum (CMhm), 8 cats (2.2%) tested positive for Mycoplasma haemofelis (Mhf), and 2 cats (0.6%) tested positive for Candidatus Mycoplasma turicensis (CMt). Co-infection with CMhm, and Mhf was observed in 7 cats (1.9%). One cat (0.3%) showed mixed infection with CMhm, Mhf, and CMt. There were statistically significant relationships between Mycoplasma positivity and being female, living in shelter (cattery), and being over 3 years old (P < 0.05). No significant association was observed for the cat breed and sampling localities. Conclusions Current study findings revealed that hemoplasma infections are common among Iran cat populations. Considering the impact of such emerging zoonotic pathogens on the One Health, routine screenings, increasing public awareness, effective control, and prophylactic strategies for minimizing infection in cats and subsequently in human are strongly recommended.


Background
Recently, the zoonoses and emerging pathogens have received increasing attention in public and animal health [1,2].Hence, there is a need for current epidemiological data to plan for rapid identification and implementation of effective control strategies to decrease their incidence in animals and humans [3].Among domestic animals around us, cats are known to be important sources for human infection by various zoonotic pathogens [1,4].
Feline vector-borne diseases which are transmitted by a variety of vectors, such as ticks, fleas, mosquitoes, and phlebotomine sand flies are of increasing importance in both public and animal health worldwide [1,2].It is partly because of their increasing incidence, challenges in their diagnosis (e.g.long incubation periods, no pathognomonic clinical signs) and subsequently their control [5].Even if some cat habits such as self-grooming can potentially minimize the establishment of some ectoparasites e.g.ticks, their outdoor access exposes them to infected arthropod vectors and consequently to the pathogens [6].Furthermore, vertical transmission of some pathogens from persistently infected queens makes the situation even more complex especially in regards to the control of zoonotic pathogens [7].
Previous studies reported prevalence of cat hemoplasmosis in different regions of the world up to 54.3% in South Korea [23].In the Middle East countries feline hMycos have been reported from Türkiye [24,25], Egypt [26], Qatar [27], and Saudi Arabia [28].In Iran however, few studies with limited number of animals from Tehran (185 domestic cats), Kerman (142 stray cats), Tehran (52 stray cats), and Tehran (19 wild felids) have been conducted [29][30][31][32].Therefore, the purpose of the current study was to provide a comprehensive information about the prevalence and molecular characterization of hMyco in apparently healthy cats from six Iranian provinces with different climates.In addition, potential risk factors associated with hemoplasma infections in cats were assessed.

Sequencing and phylogenetic analysis
BLAST analysis of six CMhm nucleotide sequences obtained in this study showed > 99% similarity with previously GenBank entries for cat (Felis catus) from Iran (HE804776, MW555992), Persian leopard (Panthera pardus) from Iran (KU852586, KU85258), European wildcat (Felis silvestris) from Germany (ON202711), also domestic cat from Chile (MN543623) and South Korea (KY432679).Nucleotide sequences of Mhf isolated from a cat of Kermanshah showed 99% similarity with the bacteria in cats from Iran (MT428555), Thailand (KJ858515) and Brazil (OQ397119).BLAST analysis also showed > 98.5% identity of CMt isolated from a cat in Hamedan with GenBank deposited entries of cats from South Korea (KY046312), Italy (KR905460) and Taiwan (JQ689950).
The constructed Maximum Likelihood (ML) tree based on the partial 16 S rRNA gene sequences of Mycoplamsa spp.showed that all sequences herein detected, clustered in well-supported clades with other known and previously reported hMycos (Fig. 2).Representative sequences of hMyco detected in this study were deposited in the GenBank® database under the accession numbers OQ421226, OQ435906, OQ417618, OQ435905 for CMhm, OR581285 for Mhf, OR581286 for CMt.

Risk factors analysis
Statistically significant relationships were observed between PCR positivity of Mycoplasma spp.(i.e.genusspecies primers) and being female, living in shelter, and being older than 3 years (P < 0.05).In addition, positivity was more prevalent in Domestic Shorthair breed (16.55%) and in cats of Khorasan Razavi (25.96%).No significant association were observed in cats for breed and sampling areas (Table 1).

Discussion
This first comprehensive epidemiological survey on apparently healthy domestic cats from Iran shows silent circulation of all three feline-infective hemotropic Mycoplasma species in different regions of the country implying its neglected health issues for cats and also the public.Infection of 15.5% of examined cats with at least one hemotropic Mycoplasma species is lower than previous studies in Iran i.e. 22-32.7% in Tehran [29,31,32] and 18.3% in Kerman [33], also reports from Western Asian and North African countries e.g.19.3% in Türkiye [25], 27.2% in Saudi Arabia [28] and 20% in Egypt [26], but higher that some other reports e.g.5.9% in Qatar [27] and 11.4% [24] in Türkiye.However, the molecular prevalence of hemoplasmas in cats in this survey was similar to studies from Germany (15.6%) [34], East and Southeast Asia (16.13%) [26], Brazil (14.6%) [35], Scotland (14.3%) [36], and UK (14%) [37].This difference in prevalence can be due to environmental and climatic factors, veterinary care, cat populations health status and living condition, availability of arthropod vectors, and methodology e.g.conventional vs. real-time PCR [12,22,36,38,39].
Co-infection with more than one species was observed in eight cats (2.2%), seven (1.9%) with CMhm + Mhf, and one with CMhm + Mhf + CMt.Similarly, multiple infection with the same two [39,45] and three [25] pathogens detected in this study were reported suggesting co-transmission of feline hemoplasmas.
In the present study, living conditions, and age were significantly associated with hemoplasma infection.The older cats were infected more often than younger ones which is in line with previous studies [22,31,34,45], and could be due to longer exposure time to pathogen and increasing risk of acquiring chronic subclinical infection over their lifetime [38,42].The significantly higher prevalence of hemoplasmas infection in shelter cats in this study is also similar to that reported in previous studies, where it has been showed that shelter animals are at higher risk of hemoplasma due to their behavior (e.g., fighting or biting), higher prevalence of ectoparasitic infestations in shelter cats, and below-standards sanitation of these shelters [5,12,[46][47][48].Finding of more positive females cats however, is in contrast with some studies that reported males are more likely to be infected with hemoplasmas [22,41,45,49] possibly because most of the female cats (32.6%) in this study were older than three years.Furthermore, in the present study, in line with previous investigations reporting that there was no significant association between hemoplasmosis and the breed of cats [7,12,50] breed was not a risk factor, although Domestic Shorthair cats displayed a higher infection rate (16.55%) than other breeds which again can be due to their proportion (41.82%) in the study.
The major limitations of the present study were the lack of information about the clinical history of the sampled cats especially antibiotic therapy e.g. with doxycycline and enrofloxacin which are known to control the infection.Furthermore, we could only test low number of pet cats and sequence limited samples because of resources.For future studies, inclusion of hematology and biochemistry profile of cats in data analysis, investigation of feline

Conclusion
This first comprehensive molecular prevalence study in domestic cats from Iran showed silent circulation of all three feline-infective hemotropic Mycoplasma species in different regions of the country implying its neglected health issues for cats and also the public.Considering the impact of such emerging and zoonotic pathogens on the One Health, routine screenings, increasing public awareness, effective control and prophylactic strategies for minimizing infection in cats and subsequently in human are strongly recommended.

Sample collection and study area
From December 2018 to February 2023, a total of 361 blood samples were collected from cephalic or saphenous vein of cats residing in six provinces of Iran with different climates namely Khorasan Razavi in the northeastern (n = 104; cold semi-arid), Kermanshah in the west (n = 85; warm and temperate), in the west Hamedan (n = 52; cold semi-arid), Kerman (n = 46; cold desert climates) in the southeast, Tehran in the central-north (n = 45; cold semiarid), and Yazd in the center (n = 29; hot and arid)(Fig.3).Blood samples were collected in sterile labeled tubes containing Ethylenediaminetetraacetic acid (EDTA) anticoagulant and stored at -70 ºC until DNA extraction.Animal data obtained at the time of sampling included gender, breed, age, and living conditions (Table 1).

Genomic DNA extraction and PCR amplification
Genomic DNA was extracted from 200 µL of the blood samples using FavorPrep™ Blood Genomic DNA Extraction Mini Kit (Favorgen, Pingtung, Taiwan) according to the manufacturer's instruction and stored at − 20 ºC until further analysis.
The conventional PCR (cPCR) assays were performed by universal Mycoplasma spp.primers targeting the partial sequence of the 16 S rRNA gene [51].Subsequently, identification of CMhm, Mhf, and CMt in PCR-positive samples was performed using a panel of three speciesspecific primers in cPCR [22].PCR reactions were performed in a 25 µL volume reaction mixture containing  [52], and phylogenetic tree was constructed using the maximum likelihood method [53], including 1000 bootstrap replicates.

Statistical analysis
Frequency analysis was performed using exact binomial 95% confidence intervals (CIs) for three Mycoplasma spp.and cPCR results.Possible associations between hemoplasmosis and potential risk factors including city, gender, breed, age, and living condition were assessed by Fisher's exact or Chi squared tests using the IBM SPSS Statistics software version 26 programs for Windows (IBM Corp, Armonk, NY).P value < 0.05 was considered as statistically significant difference.

Fig. 2
Fig. 2 Phylogenetic tree of Mycoplasma spp.isolated in this study based on a 595 bp fragment of 16 S rRNA gene using the Maximum Likelihood method.Numbers at branches represent bootstrap support levels (1000 replicates).Mycoplasma pneumoniae (GenBank: NR077056) was used as the outgroup.The CMhm, Mhf, and CMt sequences generated in this study are indicated by black circle (•), diamond (♦), and triangle (▲) symbols, respectively

Table 1
Prevalence of Mycoplasma spp. in 361 cats of Iran according to different variables

Table 2
PCR conditions and primers targeting 16 S rRNA gene of hemotropic Mycoplasma species in this study